Our laboratory is currently characterizing in vitro the functional activity of common polymorphisms of type-2 deiodinase gene. In order to achieve this goal, we have established a cell culture-based type-2 deiodinase expression system. Standard biochemistry methods are utilized for the enzymatic activity assay; protein/protein and nucleic acids/protein interactions are tested by immunoprecipitation and mobility shift assay. Very recently, a common polymorphism in the 5UTR region of the DIO2 gene (258 A/G DIO2) has been associated with a shift in the ratio of circulating T3/T4, suggesting an increased in the activity of the enzyme. Our data, consistent with the genotype/phenotype association studies, indicate that the 258 A/G DIO2 variant induces an increase in the transcription of the gene by displacing a putative repressor. Current efforts are aimed to characterize the putative repressor factor interacting with the polymorphism.. [unreadable] [unreadable] Pathophysiology of thyrotoxicosis in patients affected by McCune-Albright syndrome. The clinical observation that McCune-Albright syndrome (MAS) is associated with hyperthyroidism with a shift in the ratio of circulating T3/T4 has led to the hypothesis that this finding could be at least in part explained by a an intra-thyroidal activation of the type-2 deiodinase gene. This is in keeping with the molecular pathology of MAS, i.e. activating mutations in GNAS1 gene resulting in ligand-independent inappropriately elevated levels of intracellular cAMP. We thus speculated that activation of the cAMP-driven deiodinase type-2 gene could explain at least in part these findings. Our in vitro and ex-vivo data indicate that, consistent with our experimental hypothesis, the type-2 deiodinase is constitutively activated in MAS. Collaborative efforts with Dr. Collins (NIDCR) are aimed to develop an in vitro system to test specific inhibitors of the mutant alleles of GNAS.[unreadable] [unreadable] Pre-adipocyte differentiation and culture. We are currently in the development phase of a system of re-differentiation of human adipocytes from stromal cells obtained during adipose tissue biopsy. This system will allow to test in a controlled fashion the effects of specific genotypes on adipose tissue function relatively independent of the metabolic status of the subject at the time of the sampling.